Oligopeptide lyophilisate, their preparation and use

ABSTRACT

A novel lyophilizate and method of preparation as well as the use of the lyophilizate to treat female infertility and for gonad protection. Cetrorelix is dissolved in acetic acid 30% v/v, the solution is transferred to water and freeze dried.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 08/468,145,filed Jun. 6, 1995, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/198,037, filed Feb. 22, 1994, (now abandoned),which in turn claims priority to German Patent Application No. P4305225.8, filed Feb. 19, 1993. All applications are incorporated intheir entirety herein by reference.

The present invention relates to the preparation of a lyophilizate of apeptide and the use of the lyophilizate in the treatment of infertilityand to provide male gonad protection.

BACKGROUND OF THE INVENTION

Cetrorelix is a decapeptide with a terminal acid amide group that isused in the form of-its acetate salt. The synthesis and somepharmacological effects are described in European patent application 299402 (U.S. Pat. No. 4,800,191).

It should be possible to administer the active substance subcutaneouslyin a dose of 0.1 to 20 mg. Aqueous solutions of the decapeptide areunstable, and, therefore, autoclaving in the container used todistribute it is not possible. During conventional sterilization, usingthe prescribed conditions, the decapeptide tends to decompose. To obtainan injectable solution it was therefore necessary to develop alyophilizate.

The amount of active substance in the solution to be lyophilized is,however, so small that, in low active substance concentrations, only aloose fluff results on the glass wall of the ampoule after drying thesolution free of auxiliary substances, and this fluff is carried out ofthe vial with the stream of water vapor generated by the sublimationprocess. It is therefore necessary to use a bulking agent that forms astable cake. In high concentrations, this auxiliary substance can bedispensed with. The following auxiliary substances may be considered asbulking agents: hexitols, in particular mannitol, glucitol, sorbitol,such as D-sorbitol, dulcitol, allitol, altritol (for example D- andL-altritol), iditol (for example D- and L-iditol), their opticallyactive forms (D- and L-forms) as well as the corresponding racemates.Mannitol is used in particular, such as D-mannitol, L-mannitol,DL-mannitol, sorbitol and/or dulcitol, and, of these, D-mannitol ispreferred. The hexitol used may also be composed of a mixture of thehexitols named, for example a mixture of mannitol and sorbitol and/ordulcitol. Since dulcitol is less water soluble than, for example,mannitol, the dulcitol content in the aqueous solution should notexceed, for example, 3 percent by weight. Mannitol and sorbitol, on theother hand, can for example be mixed in any ratio.

Apart from hexitol it is also possible to add other, conventionalpharmaceutical auxiliary substances, such as amino acids, such asalanine, glycine, lysine, phenylalanine, asparaginic acid, glutaminicacid, leucine, lactose, polyvinylpyrrolidone, glucose, fructose, albuminand equivalent bulking agents. Urea and sodium chloride may also be usedas bulking agents. The total amount of such substances in the solutionwhich is used for freeze-drying, is for example 0-16.9 parts by weight,for example 0.1-7 parts by weight, based on 1 part by weight ofcetrorelix. In the finished lyophilizate the total amount of suchauxiliary substances may be up to 16.9 parts by weight, based on onepart by weight of hexitol. In detail, the amount of such auxiliarysubstances depends on the amount of hexitol present and to such anextent that the total amount of hexitol and such other auxiliarysubstances in the finished lyophilizate may not be more than a maximumof 17 parts by weight, based on 1 part by weight of cetrorelix. If only0.1 part by weight of hexitol is present in the lyophilizate, it is thuspossible to have up to 16.9 parts by weight of other auxiliarysubstances; if, for example, 8.5 parts by weight of hexitol are present,the amount of other auxiliary substances may for example be up to 8.5parts by weight, based on 1 part by weight of cetrorelix.

It was, however, found, during development work on the lyophilizate,that the active substance behaves in a widely variable and unpredictablemanner during processing. The first batches gave good results, but itsoon transpired that difficulties occurred during sterile filtration andfaulty batches resulted.

It is known from the literature, for example from Powell, M. F.;Pharmaceutical Research, 1258-1263 (8)1991; Dathe, M: Int. J. PeptideProtein Res. 344-349 (36) 1990; Szejtli, J.: Pharmaceutical TechnologyInternational 16-22, 1991 that oligopeptides, particularly those withterminal acid amide function, tend to form gels. During sterilefiltration this is apparent from the speed of filtration, indeed theincreased viscosity of such solutions can often already be detectedorganoleptically. A gelatinous layer remains on the sterile filter. Itis then no longer possible to prepare a medication with an exactly andreproducibly defined active substance content.

Table 1 lists various results of the first 11 batches.

The active substance contents fluctuate between 100% and 36%.

TABLE 1 Cetrorelix acetate Batch Dosage Active substance content % 1 100μg 100 2 500 μg 100 3 500 μg 90 4 500 μg 36 5 500 μg 100 6 500 μg 85 7 1mg 80 8 1 mg 100 9 2 mg 100 10 2 mg 80 11 2 mg 100

To avoid this gel formation, the literature lists the followingadditives which may be tried out on an experimental basis:

Organic solvents may be considered, for example acetonitrile, n-butanol,tertiary butanol, ethanol, isopropanol, octanol and benzyl alcohol. Itis also possible to use salts and buffer solutions, such as acetatebuffer, citrate buffer, sodium chloride, sodium phosphate, sodium EDTA,sodium bicarbonate, phosphate buffer, guanidine acetate, urea.

Polymers may also be used, such as gelatin, polyethylene glycol 600,hydroxyethyl starch, polyvinylpyrrolidone, polyvinyl alcohol. The use ofamino acids, for example alanine, glycine, lysine, phenylalanine,asparaginic acid, glutaminic acid and leucine has also been described.Acids that were used were citric acid, caprylic acid, octanoic acid,hydrochloric acid, sulphuric acid and acetic acid. Physiologicallyacceptable surfactants that may be used are benzalkonium chloride, cetylalcohol, bile acids, lecithins, polysorbates, Spans® and Pluronics®.

Carbohydrates and cyclodextrins such as glucose, lactose, mannitol,saccharose, alpha-, beta- and gamma cyclodextrins, hydroxypropyl-alpha-and beta-cyclodextrins, hydroxyethyl cyclodextrins and methylcyclodextrins have already been used. These auxiliary substances weretested as filtration supporting agents to prevent gel formation.

No satisfactory solution of the problem could, however, be found. Onlyacidification with acetic acid showed partial success. Here, too, itwas, however, always necessary to accept high filtration losses.

SUMMARY OF THE INVENTION

It was then surprisingly found that peptides having 3-15 amino acidssuch as cetrorelix can be easily dissolved in 30% volume/volume aceticacid. The solution is then diluted to a final concentration of 3%peptide, e.g., cetrorelix, with water for injection purposes andmannitol is added. Although it is stated in the literature that theterminal amide group hydrolyzes easily in acid medium, this was notfound in the case of cetrorelix. Solutions prepared according to thismethod caused no difficulties during filtration. The correct amounts ofactive substance were always found.

Thus, in accordance with the present invention, a peptide which contains3-15 amino acids is dissolved in acetic acid to form a solutioncontaining 100-10,000 parts by weight of acetic acid for each part ofpeptide, the solution is transferred to water, and the resultingsolution is lyophilized.

The filtration speed of the acetic acid solution attains values thatensure satisfactory production sequences. A general process for sterilelyophilization described in pages 557-559 of Sucker, Fuchs and Speiser(Publishers) “Pharmazeutische Technologie” 2nd edition 1991,Thieme-Verlag, Stuttgart-N.Y. A further description of the lyphilizationprocess used is given in German published specification (DOS) 37 35 614(U.S. Pat. No. 5,204,335).

The lyophilizate is used in the treatment of female sterility. Onetherapeutic process has hitherto consisted in stimulating folliclematuration using human menopause gonadotrophin and then triggeringovulation by administering human chorion gonadotrophin. The ovulationtriggered thereby occurred 32 hours later. The resulting ova areavailable for in vitro fertilization.

A disadvantage of this treatment with agonists is the fact that up to 10follicles mature during the stimulation phase. This elevated folliclematuration leads to hormone level peaks in the LH. These peaks result inan early stage of follicle maturation and ovulation at an unpredictedpoint in time. This impaired ovulation occurs in about 25% of treatedcases and is a disadvantage since the cycle that displays disturbedovulation of this kind cannot be used for the collection of ova and theentire treatment has to be repeated about 1 month later.

Another disadvantage of the conventional simulation treatment and theuse of LHRH agonists in order to avoid premature LH-peaks is the longtreatment duration of 4 weeks which is needed to achieve satisfactorysuppression. The agonists continue to display a hyperstimulationsyndrome in 1-2% of cases in which the follicle cells hypertrophy. Therisk of hyperstimulation is particularly great in the case of polycysticovaries. The hyperstimulation syndrome is a severe side effect which canlead to fatalities.

It has now been found that the antagonist cetrorelix displays thefollowing advantages in this treatment:

Treatment with cetrorelix over 5 days is sufficient to achieve totalsuppression. The premature LH peaks cannot arise and the frequency ofhyperstimulation syndrome should be reduced. In addition, less HMG isused in the 2nd phase of therapy, the ovulation triggering phase. Thisgives this in-vitro fertilization treatment a not inconsiderable costadvantage. In-vitro fertilization is, for example, used when a tubeanomaly is present. To perform this treatment it is necessary toprecisely monitor the cycle and to establish the time of ovulation asprecisely as possible. This has hitherto only been achieved to a limitedextent since preovulatory LH increase often occurred too early due tosimulation with HMG/HCG, or was not maintained for a sufficiently longperiod. Avoidance of this premature increase is, however, of criticalimportance for the success-of-the treatment in order to preciselydetermine the time of fertilization. This reduces the physical andmental burden on the patient and makes optimum use of hospitallogistics. To achieve this objective with great reliability it isnecessary to suppress endogenous hormone production (LH-FSH, oestradiol)as completely as possible in order to simultaneously stimulate folliclematuration through administration of exogenous gonadotrophins (HMG/HCG)and to monitor the hormone status at any time. It is only when asufficiently large number of follicles have been achieved (4-6), havingapproximately the same degree of maturation, that ovulation is triggeredby administering an HCG bolus injection.

Use of an antagonist makes treatment substantially more successful andsafer for the patient.

Another area of use of the cetrorelix lyophilizate according to thepresent invention is to protect the gonads in male patients. Malepatients are pre-treated with cetrorelix lyophilizate and the activityof the gonads is reinforced. As a result, other harmful noxious agents,such as radiation therapy or treatment with cytostatics, have no or onlya small possibility of affecting the sensitive tissue of the gonads.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The following Examples illustrate the invention.

EXAMPLE 1

Approx. 1.5 liters of water for injection purposes are prepared in asuitable glass vessel. 210 g water for injection purposes are preparedin another glass vessel and 91.17 g acetic acid are added. The amount ofcetrorelix acetate calculated (1.62-1.695 g, depending on the content ofthe batch used) is dissolved in the prepared 30% acetic acid withstirring. This solution is transferred to the glass vessel with 1.5liters of water for injection purposes, 82.2 g mannitol are added,dissolved and made up to 3039 g with water for injection purposes.

In-process checks: pH value: 2.5-3.0 Density: 1.0213-1.0225 g/cm³ at 20°C. Refractive Index: 1.335-1.345 at 589 nm at 20° C.

The solution is sterilized by filtration through an appropriate membranefilter (pore size 0.2 μm) under aseptic conditions. 100 ml firstrunnings should be discarded. The filters should be sterilized withsuperheated steam before sterile filtration. Cetrorelix freeze-driedsolution should be protected from recontamination during storage.

The solution is immediately filled into colorless injection bottles DIN2R, hydrolytic class I under aseptic conditions and provided withsterile freeze-drying stoppers. The nominal filling amount is 2.0ml=2.026 g.

The 2 ml injection bottles were rinsed in an injection bottle washingmachine, dried with hot air and sterilized. The cleaned, freeze-dryingstoppers were autoclaved. The closed injection bottles were transferredto a freeze-drying installation and frozen at a plate temperature of−40° C. Drying was carried out using a drying program with a platetemperature of −40° C. rising to +20° C. The installation is thenflooded with sterile nitrogen, the bottles are closed in theinstallation and the stoppers secured with crimped caps.

The injection bottles are checked visually for faulty closures and outerfaults. Faulty injection bottles are removed and destroyed.

Cetrorelix lyophilizate 1 mg is a white, solid, freeze-dried cake in acolorless 2 ml injection bottle which is closed with gray freeze-dryingstoppers and yellow flip-off crimped caps.

EXAMPLE 2

Nonapeptide (Bombesin-antagonist)

420 g water for injection purpose are prepared in a suitable vessel and121.56 g acetic acid are added. The amount of the nonapeptide (about3.783 g, depending on the content of the batch used) is dissolved in theprepared 20% acetic acid and with stirring. 82., 2 niannitol are addedand dissolved. This solution is sterilized by filtration through anappropriate membrane filter (pore size 0.2 μm) under aseptic conditions.The same membrane filter is used for the water for injection purpose tomake up the solution to 3064 g. The filters should be sterilized withsuperheated steam.

In-process checks: pH value: 2.5-3.0 Density: 1.0232-1.0252 g/cm³ at 20°C. Refractive Index: 1.334-1.344 at 589 nm at 20° C.

The solution should be protected from recontamination during storage.The solution is filled in to sterile colorless injection bottles DIN 2R, hydrolytic class I under aseptic conditions and provided with sterilefreeze-drying stoppers. The nominal filling amount is 1.0 ml=1.022 g.

The 2 ml injection bottles were rinsed in an injection bottle washingmachine, dried with hot air and sterilized. The cleaned freeze-dryingstoppers were autoclaved. The injection bottles were transferred to afreeze-drying installation and frozen at a plate temperature of −40° C.

Drying was carried out using a drying programme with a plate temperatureof −40° C. rising to +20° C. The installation is then flooded withsterile nitrogen, the bottles are closed in the installation and thestoppers are sealed with crimped caps.

The injection bottles are checked visually for faulty closures and outerfaults. Faulty injection bottles are removed and destroyed.

The lyophilisate of the nonapeptide (1 mg) is a white, solid,freeze-dried cake in a colorless 2 ml injection bottle which is closedwith grey freeze-drying stoppers and flip-off crimped caps.

EXAMPLE 3

Tripeptide (Protirelin)

143.5 g water for injection purpose are prepared in a suitable vesseland 61.5 g acetic acid are added. The amount of the Protirelin acetatecalculated (equivalent to 800 mg of the peptide base) is dissolved withstirring. This solution is transferred to another vessel withapproximately 1 l water for injection purpose. 109.6 g mannitol areadded, dissolved and made up to 2048 g with water for injectionpurposes.

In-process checks: pH value: 2.5-3.0 Density: 1.0235-1.0255 g/cm³ at 20°C. Refractive Index: 1.336-1.348 at 589 nm at 20° C.

The solution is filled in to sterile colorless injection bottles DIN 2R, hydrolytic class I under aseptic conditions and provided with sterilefreeze-drying stoppers. The nominal filling amount is 1.0 ml=1.024 g.

The 2 ml injection bottles were rinsed in-an injection bottle washingmachine, dried with hot air and sterilized. The cleaned freeze-dryingstoppers were autoclaved. The injection bottles were transferred to afreeze-drying installation and frozen at a plate temperature of −40° C.

Drying was carried out using a drying programme with a plate temperatureof −40° C. rising to +20° C. The installation is then flooded withsterile nitrogen, the bottles are closed in the installation and thestoppers are sealed with crimped caps.

The injection bottles are checked visually for faulty closures and outerfaults. Faulty injection bottles are removed and destroyed.

The Protireline lyophilizate (0.4 mg) is a white, solid, freeze-driedcake in a colorless 2 ml injection bottle which is closed with greyfreeze-drying stoppers and flip-off crimped caps.

EXAMPLE 4

Tetradecapeptide (Somatostatin)

245 g water for injection purpose are prepared in a suitable vessel and61.5 g acetic acid are added. The amount of somatostatine acetatecalculated (0.52-0.66 g, dependent on the content of the batch used) isdissolved with stirring. This solution is transferred to another vesselwith approximately 1 l water for injection purpose. 109.6 g mannitol areadded, dissolved and made up to 2049 g with water for injectionpurposes.

In-process checks:

pH value: 2.5-3.0

Density: 1.0232-1.0252 g/cm³ at 20° C.

Refractive index: 1.336-1.348 at 589 nm at 20° C.

The solution is filled in to sterile colorless injection bottles DIN 2R, hydrolytic class I under aseptic conditions and provided with sterilefreeze-drying stoppers. The nominal filling amount is 1.0 ml=1.024 g.

The 2 ml injection bottles were rinsed in an injection bottle washingmachine, dried with hot air and sterilized. The cleaned freeze-dryingstoppers were autoclaved. The injection bottles were transferred to afreeze-drying installation and frozen at a plate temperature of −40° C.

Drying was carried out using a drying programme with a plate temperatureof −40° C. rising to +20° C. The installation is then flooded withsterile nitrogen, the bottles are closed in the installation and thestoppers are sealed with crimped caps.

The injection bottles are checked visually for faulty closures and outerfaults. Faulty injection bottles are removed and destroyed.

The lyophilizate (0.25 mg somatostatine acetate) is a white, solid,freeze-dried cake in a colorless 2 ml injection bottle which is closedwith grey freeze-drying stoppers and flip-off crimped caps.

1. A method of treating female infertility which comprisesadministering, to a female undergoing controlled ovarian stimulation, aneffective amount of pharmaceutical composition comprising a lyophilizateof a certrorelix solution containing 100-10,000 parts by weight ofacetic acid per part of dissolved cetrorelix, thereby preventingpremature ovulation.
 2. The method according to claim 1, wherein thelyophilizate further comprises a bulking agent.